Evaluation of the effect of tyrothricin on beta-hemolytic streptococci in salva. Part I: The effect of salvia upon bacteria. Part II: Effect of tyrothricin on the New York 5 strain of Streptococcus pyogenes in saliva

Abstract

Part II of thesis by Brancato, Noyes, and Swift. Part I of thesis by Swift. Thesis (M.A.)--Boston UniversityThe antibacterial effect of saliva has been known for many years. Still the exact nature of the antagonistic action of saliva upon bacteria is as yet unsettled. Most workers agree, however, that the salivary bacterial inhibitory action is brought about in at least six ways: The first antibacterial effect is changes in pH, which affect the growth of oral organisms. Furthermore, this change in pH is dependent on diet and on the type of organisms in the oral cavity. The second is the mechanical factors involved, for saliva not only flushes bacteria from the mouth, but dilutes the number of organisms as well. The third is the antibacterial action of the cellular components in saliva. The leukocytes in saliva have a phagocytic action, and the non-phagocytic epithelial cells slough off in sheets, carrying with them thousands of organisms which have lodged in the partially turned edges of the necrotic cells . The fourth antibacterial action is ascribed to the presence of immune bodies in the saliva which lyse or agglutinate the oral bacteria. The fifth is the presence of oral bacteria which are antagonistic to new invaders. And the sixth is the presence of enzymes that lyse some oral bacteria or alter their cell membranes thereby inhibiting further growth. In recent years a great deal of investigation has been made to ascribe the enzymatic effect as the chief antibacterial agent in saliva; however, contradictory work has been done to try to attribute the chief antibacterial action of salivary cocci. Indeed the antibacterial effect of saliva is not always present, for the bacteriostatic effect of saliva is variable from day to day and from individual to individual. The only way of reducing the number of oral bacteria is to add to the saliva an antibiotic. Tyrothricin was used. In an attempt to delineate the range of concentration of tyrothricin per ml. effective against the New York 5 strain of Streptococcus pryogenes in saliva, this experiment was carried out. It was molded after the unpublished work of Belding concerning the effect of tyrothricin on the Oxford Strain of Staphylococcus aureus in saliva. The required inoculum of approximately one million organisms per ml was obtained by growing cultures of the streptococci under uniform conditions and setting up a table of the absorbances and viable cell counts, from which dilution factors for further cultures could be estimated. Controls were set up for determining possible inhibition of tyrothricin and/or test organisms by the various diluting fluids including saliva. Final concentrations per ml of 10, 25, 50, 75, and 100 µg of tyrothricin integrated with saliva and an approximated number of streptococci were plated out after 30 and 60 minutes exposure periods and were counted after 24 and 48 hours of incubation at 37°C. Whereas 1 µg per ml of tyrothricin reduced markedly the number of streptococci suspended in water during a 30 minute exposure period and 10 µg per ml, under similar conditions, caused complete inhibition, 10 µg per ml of the antibiotic was ineffective against this test organism suspended in saliva during a 30 minute exposure period but caused about an 80 per cent reduction in viable organisms during 60 minutes exposure. The length of the exposure period necessary for effective inhibition varied inversely with the concentration of tyrothricin per ml, 100 µg per ml causing a 98 per cent reduction of viable organisms during an exposure period of 1 minute. For the 30 minute exposure period, the quantity of tyrothricin effective against this strain of streptococci mixed in saliva would fall in the 10 µg - 25 µg per ml range and for shorter exposure periods, the concentration per ml would have to be greater. Cultures completely negative during 24 hours incubation at 37°C, showed a typical growth during 48 hours. This is considered indicative of the bacteriostatic action of tyrothricin which, prolonged, resulted in the death of large numbers of the streptococci. The results which were obtained in these experiments serve chiefly to point out the way for further work and to form a basis for the general conclusions listed below: 1. The action of tyrothricin on bacteria is inhibited by saliva to a large degree. 2. The minimal amounts of tyrothricin necessary to produce complete inhibition of growth of Streptococcus pyogenes in saliva is between 25 and 50 µg per ml acting for 30 minutes. 3. There is an effective reduction of Streptococcus pyogenes in saliva by concentrations of tyrothricin between 10 and 25 µg per ml acting for 30 minutes. 4. Tyrothricin acts immediately upon contact with Streptococcus pyogenes. 5. The action of tyrothricin on Streptococcus pyogenes in saliva is apparently bacteriostatic and not of a permanent nature as manifested by growth of atypical colonies during 48 hours incubation. 6. Tyrothricin above a concentration of 50 µg per ml had a definite reducing effect on the bacterial population of this saliva. 7. Saliva also has a bactericidal or bacteriostatic (or both) action against Streptococcus pyogenes